Use of recombinant inhibitor from Erythrina caffra for purifying serine proteases

ABSTRACT

Process for purifying serine proteases from a protein mixture by binding the serine protease to an immobilized polypeptide with the activity of an inhibitor DE-3 from Erythrina caffra, removing unbound components from the protein mixture, detaching the serine protease from the inhibitor and separating the immobilized inhibitor from the soluble serine protease and isolating serine protease which is characterized in that a polypeptide is used as the polypeptide which is the product of a prokaryotic or eukaryotic expression of an exogenous nucleic acid. This inhibitor is distinguished by an improved specific activity and is particularly suitable for the purification of plasminogen activators such as tissue plasminogen activators (t-PA and derivatives).

This is a U.S. national stage application of PCT/EP95/00926 filed onMarch 13, 1995.

The invention concerns an improved process for purifying serineproteases using a recombinant inhibitor DE-3 from Erythrina caffra.Immobilized trypsin inhibitors from Erythrina (ETI) are effectivereagents for the affinity chromatographic purification of serineproteases and in particular of plasminogen activators (C. Heussen, J.Biol. Chem. 259 (1984) 11635-11638), β-trypsin, α-chymotrypsin andthrombin (S. Onesti et al., J. Mol. Recogn. 5 (1992) 105-114). Thesetrypsin inhibitors have been known for a long time (C. Heussen,Haemostasis 11 (1982) P47 (Supplement); F. J. Joubert, Phytochemistry 21(1982) 1213-1217; F. J. Joubert, Int. J. Biochem. 14 (1982) 187-193).

The inhibitor DE-3 from E. caffra is particularly suitable for thepurification of plasminogen activators (F. J. Joubert in Thrombosis andHaemostasis 57 (1987) 356-360). The complete amino acid sequence of thisinhibitor is also described in this publication. DE-3 can be isolatedand purified from the seeds of E. caffra (F. J. Joubert, Int. J.Biochem. 14 (1982) 187-193).

A recombinant ETI is described by Teixeira et al., Biochimica etBiophysica Acta 1217 (1994) 16-22, whose specific inhibitory activityfor tissue plasminogen activator is 1.7×10⁹ U/mmol. In contrast thespecific inhibitory activity of natural ETI is 1.94×10⁹ U/mmol.

A similar situation applies to the inhibitory activity towards trypsin(2.63×10¹² /3.21×10¹²). Thus the specific inhibitory activity towardstrypsin and tissue plasminogen activator of recombinant ETI preparedaccording to Teixeira is 20% less than the activity of natural ETI.

Recombinant ETI is obtained by expression according to Teixeira andpurified by ammonium sulfate precipitation (80% saturation), dialysisagainst water and a cyanogen bromide cleavage in which the N-terminalsequence including the methionine is cleaved off. It is subsequentlychromatographed on an affinity chromatography column (Sephadex G50).

The object of the invention is to improve the effectiveness of processesfor the purification of serine proteases using ETI.

The invention concerns a process for the purification of serineproteases from a protein mixture by binding the serine protease to animmobilized polypeptide which has the activity of an inhibitor DE-3 fromErythrina caffra, removing the unbound components from the proteinmixture, detaching the serine protease from the inhibitor, separatingthe immobilized inhibitor from the soluble serine protease and isolatingthe serine protease which is characterized in that a polypeptide is usedas the polypeptide which is the product of a prokaryotic or eukaryoticexpression of an exogenous nucleic acid (preferably DNA) and is purifiedchromatographically by means of an anion exchanger, cation exchanger ora nickel chelate column. surprisingly it was found that the recombinantpolypeptide produced according to the invention which has the activityof an inhibitor DE-3 from Erythrina caffra has a substantially increasedspecific affinity towards serine proteases compared to inhibitor DE-3from E. caffra isolated from natural sources.

The "activity" of an inhibitor DE-3 from E. caffra is essentially to beunderstood as its specific inhibitory activity towards serine proteasesin particular to tissue plasminogen activators. In this case thespecific inhibitory activity of the inhibitor is at 1.07 U/mg or morewith regard to trypsin. The inhibition is achieved by binding betweeninhibitor and serine protease.

The process according to the invention is particularly advantageous forpurifying plasminogen activators such as tissue plasminogen activators(t-PA) and derivatives (e.g. mutations and deletions) thereof. t-PA andderivatives are described for example in EP-B 0 093 619, U.S. Pat. No.5,223,256, WO 90/09437 and T. J. R. Harris, Protein Engineering 1 (1987)449-458.

The production of recombinant inhibitors can be carried out according tomethods familiar to a person skilled in the art.

For this a nucleic acid (preferably DNA) is firstly prepared which isable to produce a protein which possesses the activity of the inhibitorDE-3. The DNA is cloned into a vector that can be transferred into ahost cell and can be replicated there. Such a vector contains operatorelements in addition to the inhibitor sequence which are necessary forthe expression of the DNA. This vector which contains the inhibitor DNAand the operator elements is transferred into a vector that is able toexpress the DNA of the inhibitor. The host cell is cultured underconditions that allow the expression of the inhibitor. The inhibitor isisolated from these cells. During this process suitable measures areundertaken to ensure that the activator can assume an active tertiarystructure in which it exhibits inhibitor properties.

In this connection it is not necessary that the inhibitor has the exactamino acid sequence corresponding to SEQ ID NO: 2. Inhibitors are alsosuitable which have essentially the same sequence and which arepolypeptides with the activity (capability of binding to serineproteases, in particular to t-PA) of an inhibitor DE-3 from Erythrinacaffra. It has turned out that homology of the amino acid sequence of80% is advantageous, preferably of 90%. However, the amino acid sequenceSEQ ID NO:2 is preferably used which in the case of expression inprokaryotic host cells, but not after eukaryotic expression, contains aN-terminal methionine.

The invention in addition concerns a nucleic acid which is essentiallyidentical to the nucleotides 9 to 527 of SEQ ID NO: 1 and codes for apolypeptide with the activity of an inhibitor DE-3 from Erythrinacaffra, or a nucleic acid which codes for the same polypeptide withinthe scope of the degeneracy of the genetic code. A DNA is preferred andin particular a DNA of the sequence 9 to 527 of SEQ ID NO: 1. For theexpression in eukaryotic or prokaryotic host cells, the nucleic acidcontains at its 5' end the eukaryotic or prokaryotic transcription andtranslation signals which are familiar to a person skilled in the art.

The nucleic acid sequence of the inhibitor is preferably identical tothe nucleotides 9 to 527 of SEQ ID NO:1. However, modifications may bemade in order to facilitate the production of the vectors or to optimizeexpression. Such modifications are for example:

Modification of the nucleic acid in order to introduce variousrecognition sequences of restriction enzymes in order to facilitateligation, cloning and mutagenesis steps

Modification of the nucleic acid in order to incorporate preferredcodons for the host cell

Extension of the nucleic acid by additional operator elements in orderto optimize expression in the host cell.

The inhibitor is preferably expressed in microorganisms such as E. coli.It is, however, also possible to carry out the expression in eukaryoticcells such as yeast, CHO cells or insect cells.

For this, biological functional plasmids or viral DNA vectors are usedwhich essentially contain the nucleotides 9 to 527 of SEQ ID NO:1 or anucleic acid which codes for the same polypeptide within the scope ofthe degeneracy of the genetic code. Prokaryotic or eukaryotic host cellsare stably transformed or transfected with such vectors.

The expression vectors must contain a promoter that allows theexpression of the inhibitor protein in the host organism. Such promotersare known to a person skilled in the art and are for example the lacpromoter (Chang et al., Nature 198 (1977) 1056), trp (Goeddel et al.,Nuc. Acids Res. 8 (1980) 4057), .sup.λ PL promoter (Shimatake et al.,Nature 292 (1981) 128) and T5 promoter (U.S. Pat. No. 4,689,406).Synthetic promoters such as for example the tac promoter (U.S. Pat. No.4,551,433) are also suitable. Coupled promoter systems such as forexample the T7 RNA polymerase/promoter system (Studier et al., J. Mol.Biol. 189 (1986) 113) are also suitable. Hybrid promoters from abacteriophage promoter and the operator region of the microorganism(EP-A 0 267 851) are also suitable. In addition to the promoter it isalso necessary to have an effective ribosome binding site. In the caseof E. coli this ribosome binding site is denoted Shine-Dalgarno (SD)sequence (Shine et al., Nature (1975) 25434; J. Sambrook et al.,"Expression of cloned genes in E. coli" in Molecular Cloning: Alaboratory manual (1989) Cold Spring Harbor Laboratory Press, USA).

In order to improve the expression it is possible to express theinhibitor protein as a fusion protein. In this case a DNA which codesfor the N-terminal part of an endogenous bacterial protein or anotherstable protein is usually fused to the 5' end of the sequence coding forthe inhibitor protein. Examples of this are lacZ, trpE.

After expression the fusion proteins are preferably cleaved with enzymes(e.g. factor Xa) (Nagai et al., Nature 309 (1984) 810). Further examplesof cleavage sites are the IgA protease cleavage site (WO 91/11520) andthe ubiquitin cleavage site (Miller et al., Bio/Technology 7 (1989)698).

The recombinant protein that is firstly obtained as inactive inclusionbodies can be converted into a soluble active protein by methodsfamiliar to a person skilled in the art. For this the inclusion bodiesare for example solubilized with guanidine hydrochloride or urea in thepresence of a reducing agent, reduced, the reducing agent is removede.g. by dialysis and preferably renatured using a redox system such asreduced and oxidized glutathione.

Such methods are described for example in U.S. Pat. No. 4,933,434, EP-B0 241 022 and EP-A 0 219 874.

It is also possible to secrete the proteins from the microorganism asactive proteins. For this a fusion protein is preferably used which iscomposed of the signal sequence that is suitable for the secretion ofproteins in the host organisms used (U.S. Pat. No. 4,336,336) and thenucleic acid which codes for the inhibitor protein. In this case theprotein is either secreted into the medium (in the case of gram-positivebacteria) or into the periplasmatic space (in the case of gram-negativebacteria). It is expedient to introduce a cleavage site between thesignal sequence and the sequence coding for the inhibitor which enablesthe cleavage of the inhibitor protein either during processing or in anadditional step. Such signal sequences are for example ompA (Ghrayeb etal., EMBO J. 3 (1984) 2437) and phoA (Oka et al., Proc. Natl. Acad.

Sci. USA 82 (1985) 7212).

The vectors in addition contain terminators. Terminators are DNAsequences that signal the end of a transcription process. They areusually characterized by two structural features: a reversed repetitiveG/C-rich region which can intramolecularly form a double helix and anumber of U (or T) residues. Examples are the trp attenuator andterminator in the DNA of the phage fd and rrnB (Brosius et al., J. Mol.Biol. 148 (1981) 107-127).

In addition the expression vectors usually contain a selectable markerin order to select transformed cells. Such selectable markers are forexample the resistance genes for ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracyclin (Davies et al., Ann.Rev. Microbiol. 32 (1978) 469). Selectable markers which are alsosuitable are the genes for substances essential for the biosynthesis ofsubstances necessary for the cell such as e.g. histidine, thryptophanand leucine.

Numerous suitable bacterial vectors are known. For example vectors havebeen described for the following bacteria: Bacillus subtilis (Palva etal., Proc. Natl. Acad. Sci. USA 79 (1982) 5582), E. coli (Aman et al.,Gene 40 (1985) 183; Studier et al., J. Mol. Biol. 189 (1986) 113),Streptococcus cremoris (Powell et al., Appl. Environ. Microbiol. 54(1988) 655), Streptococcus lividans and Streptomyces lividans (U.S. Pat.No. 4,747,056).

In addition to prokaryotic microorganisms, it is also possible toexpress inhibitor proteins in eukaryotes (such as for example CHO cells,yeast or insect cells). Yeast and insect cells are preferred as theeukaryotic expression system. The expression in yeast can be achieved bythree types of yeast vectors (integrating YIp (yeast integratingplasmids) vectors, replicating YRp (yeast replicon plasmids) vectors andepisomal YEp (yeast episomal plasmids) vectors). Further details ofthese are described for example in S. M. Kingsman et al., Tibtech 5(1987) 53-57.

Further genetic engineering methods for the production and expression ofsuitable vectors are described in J. Sambrook et al., "Expression ofcloned genes in E. coli" in Molecular cloning: A laboratory manual(1989) Cold Spring Harbor Laboratory Press, USA).

After production, the recombinant ETI is purified chromatographically bymeans of an anion exchanger such as a Q-Sepharose® column, a cationexchanger (e.g. based on sulfopropyl) or by means of a nickel chelatecolumn as described for example in Porath, J. & Olin, B., Biochemistry22 (1983), 1621-1630. surprisingly after this purification procedure arecombinant ETI is obtained whose inhibitory activity towards serineproteases such as trypsin and tissue plasminogen activator issubstantially higher than the inhibitory activity of natural ETI.

A polypeptide prepared and purified in this manner has the activity of aDE-3 inhibitor from Erythrina caffra and is obtainable by culturingprokaryotic or eukaryotic host cells which are transformed ortransfected with an exogenous nucleic acid (preferably DNA) whichessentially corresponds to the sequence of nucleotides 9 to 527 of SEQID NO: 1 or to a nucleic acid which codes for the same polypeptidewithin the scope of the degeneracy of the genetic code, in a manner thatenables the host cells to express the polypeptide under suitablenutrient conditions and isolating the desired polypeptide which has aspecific inhibitory activity of ca. 1.07 U/mg or more (preferably 1.07to 1.8 U/mg) towards trypsin. In various lots of the recombinant ETI aspecific activity of for example 1.2, 1.5 and 1.6 U/mg was found. Thisactivity is obtained after chromatographic purification on an anionexchanger, cation exchanger or on a nickel chelate column.

The invention in addition concerns a process for the production of arecombinant polypeptide which has the activity of a DE-3 inhibitor fromErythrina caffra (ETI) by culturing prokaryotic or eukaryotic host cellswhich are transformed or transfected with an exogenous nucleic acid(preferably DNA) which essentially corresponds to the sequence ofnucleotides 9 to 527 of SEQ ID NO: 1 or to a nucleic acid which codesfor the same polypeptide within the scope of the degeneracy of thegenetic code, in a manner that enables the host cells to express thepolypeptide under suitable nutrient conditions, isolating thepolypeptide from the host cells and chromatographic purification on ananion exchanger, cation exchanger or on a nickel chelate column.

The purification of serine proteases using recombinant ETI is carriedout according to methods familiar to a person skilled in the art (cf.e.g. F. J. Joubert (1987)). For this ETI is bound covalently to a matrix(e.g. CNBr-Sepharose colum) and the protein mixture which contains theserine protease is applied to the column under neutral or weaklyalkaline conditions and a chromatography is carried out. The elution isachieved by lowering the pH to < pH 5.5 or by using buffer solutionsthat contain chaotropic agents such as e.g. KSCN. The eluate has aprotein purity of over 95% with regard to the serine protease. Forfurther use it is expedient to transfer the serine protease into thebuffer solution that is desired in each case by dialysis.

The immobilization of the inhibitor and all further steps in theprocedure for the purification of serine protease and t-PA can becarried out in an analogous manner to that for the inhibitor DE-3isolated from E. caffra. Such processes are for example described inEP-B 0 218 479, EP-B 0 112 122, U.S. Pat. No. 4,902,623. It is expedientto carry out the immobilization on an inert carrier, preferably onCNBr-Sepharose®.

The invention is described in more detail by the following examples andsequence protocols.

EXAMPLE 1 Expression of ETI in E. coli

a) Gene synthesis

A corresponding nucleic acid sequence was derived from the amino acidsequence of ETI from Erythrina caffra (Joubert and Dowdle, Thrombosisand Haemostasis 57 (3) (1987) 356-360) using the codons preferred by E.coli and prepared synthetically according to the method by Beattie andFowler (Nature 352 (1991) 548-549). In order to facilitate the cloning,a cleavage site for the restriction enzyme EcoRI was inserted at the 5'end and a cleavage site for the restriction enzyme HindIII was insertedat the 3' end. The synthesized nucleic acid was recleaved with theenzymes EcoRI and HindIII and ligated with the cloning vector pBS+(Stratagene, US, Catalogue No 211201, derivative of the fl phage andStratagene's pBS plasmid with the T3 and T7 promoter gene, ampicillinresistance gene, fl origin, ColE-1 origin, lacd gene, lacZ gene and amultiple cloning site) which previously had also been digested withEcoRI and HindIII. The ligation preparation was transformed intoEscherichia coli. The clones obtained, selected on ampicillin, wereanalysed by restriction with the enzymes EcoRI and HindIII. Theresulting clone, pBS+ETI, contains an additional EcoRI/HindIII fragmentwith a size of 539 bp and has SEQ ID NO: 1.

b) Expression vector

Plasmid pBS+ETI was recleaved with the restriction enzymes EcoRI andHindIII and the 539 bp large fragment was isolated. The expressionvector pBTacl (from Boehringer Mannheim GmbH, Catalogue No. 1081365,based on pUC8, H. Haymerle et al., Nucl. Acid Res. 14 (1986) 8615-8624)was likewise digested with the enzymes EcoRI and HindIII and the 4.6 kblarge vector fragment was isolated. Both fragments were ligated andtransformed together into E. coli (DSM 5443) together with the helperplasmid pUBS520 (Brinkmann et al., Gene 85 (1989) 109-114) whichcontains the lac repressor gene. The clones were selected on the basisof the ampicillin and kanamycin resistance mediated by the plasmids.Plasmid pBTETI obtained contains an additional EcoRI/HindIII fragmenthaving a size of 539 bp compared to the starting vector pBTac1.

DSM 3689 which already contains an I^(q) plasmid can be used in ananalogous manner instead of DSM 5443. In this case the helper plasmidpUB520 is not necessary.

c) Expression of recombinant ETI (recETI) in E. coli

In order to check the expression rate, the E. coli strain DSM 5443 wascultured with plasmids PBTETI and pUBS520 in LB medium (Sambrook et al.,Molecular Cloning (1989) Cold Spring Harbor) in the presence ofampicillin and kanamycin (50 μg/ml final concentration in each case) toan optical density (OD) of 0.6 at 550 nm. The expression was initiatedby addition of 5 mM IPTG. The culture was incubated for a further 4hours. Subsequently the E. coli were collected by centrifugation andresuspended in buffer (50 mM Tris-HCl pH 8, 50 mM EDTA); lysis of E.coli was achieved by sonification. The insoluble protein fractions(inclusion bodies) were collected by renewed centrifugation andresuspended in the above-mentioned buffer by sonification. Thesuspension was admixed with 1/4 volumes application buffer (250 mMTris-HCl pH 6.8, 0.01 M EDTA, 5% SDS, 5% mercaptoethanol, 50% glyceroland 0.005% bromophenol blue) and analysed with the aid of a 12.5% SDSpolyacrylamide gel. As a control the same preparation was carried outwith a culture of E. coli (pBTETI/pUBS520) which had not been admixedwith IPTG and applied to the polyacrylamide gel. A clear band with amolecular weight of about 22 kD can be seen in the preparation of theIPTG-induced culture after staining the gel with 0.2% Coomassie blueR250 (dissolved in 30% methanol and 10% acetic acid) and destaining thegel in a methanol-acetic acid mixture.

This band cannot be found in the preparation of the non-induced E. colicells.

EXAMPLE 2 Renaturation and purification of recETI

50 g inclusion bodies (IBs) were solubilized with 0.1 M Tris/HCl, pH8.5, 6 M guanidine, 0.1 M DTE, 1 mM EDTA (90 min at 25° C., C_(prot).=10 mg/ml) and, after adjusting the pH value to 2.5 (HCl), dialysedagainst 3 mol/l guanidine/HCl. The dialysate was centrifuged (SS34,13,000 rpm) and adjusted to C_(prot). =36.9 mg/ml by concentration overYM 10. A 1 l reaction vessel was filled with 0.1 M Tris/HCl, pH 8.5, 1mM EDTA, 1 mM GSH, 0.1 mM GSSG. The renaturation was carried out at 20°C. by a 16-fold addition of the dialysate (600 μg protein/ml buffer eachtime) at intervals of 30 min.

The renaturation yields 2.8 U/ml active ETI.

Purification of recETI

a) by means of an anion exchanger

RecETI is renatured in 0.1 M Tris/HCl, pH 8.5, 1 mM EDTA, 1 mM GSH, 0.1mM GSSG. The renaturate is diluted 1:2 with H₂, adjusted to pH 8.0 withHCl, dialysed against 50 mM Tris/HCl pH 8.0 and applied to aQ-Sepharose® column equilibrated with 50 mM Tris/HCl, pH 8.0 (5 mgprotein/ml gel). After washing the column with equilibration buffer andwith 50 mM Na₂ HPO₄ /H₃ PO4, pH 8.0 (five column volumes each time),elution is achieved with 50 mM Na₂ HPO_(4/) H₃ PO₄, pH 8.0, 0.2 M NaCl.

b) by means of a cation exchanger

Renatured ETI was adjusted to pH 4.0 by addition of HCl and dialysedagainst 50 mM NaOAc/HCl, pH 4.0 (Cross Flow). The dialysate wascentrifuged (13,000 rpm, 30 min, SS 34) and applied to a TSK-SP column(cation exchanger with sulfopropyl side groups, Merck, Germany, volume15 ml) that had been equilibrated with 50 mM NaOAc/HCl, pH 4.0. Afterwashing the column with the equilibration buffer and with 50 mMNaOAc/HCl, pH 4.0, 0.1 M NaCl, it is eluted with 50 mM NaOAc/HCl, pH4.0, 0.2 M NaCl.

The purity of the eluate was examined by SDS-PAGE and by means ofRP-HPLC.

RESULT

ETI binds to the TSK-SP column under the conditions used and can beeluted with 0.2 M NaCl. SDS-PAGE and RP-HPLC analysis result in a purityof >95%.

EXAMPLE 3 Comparison of the Specific Activity of recETI and of ETI FromSeeds of Erythrina Caffra

RecETI and ETI isolated from the seeds of E. caffra were dialysedagainst 50 mM Na₂ HPO₄ /H₃ PO₄, pH 8.0, 0.2 M NaCl and adjusted to aprotein concentration of 0.8 mg/ml. The protein concentration wasdetermined by measuring the UV absorbance at 280 nm (ε=1.46 cm² /mg).

Determination of the ETI Activity

The inhibition of trypsin by ETI is measured using N-α-benzoylethylester (BAEE) as the substrate. 40 μl trypsin solution (0.13 mg/ml 2 mMHCl) is mixed with 60 μl test buffer (0.1 M Tris/HCl, pH 8.0) and 100 μlETI solution in a quartz cuvette and incubated for 5 min at 30° C. Afteraddition of 800 μl BAEE solution (20 mg BAEE×HCl/100 ml test buffer) theincrease in absorbance/min is determined at 253 nm.

The ETI activity is determined according to the following formula:

    U/ml= 1-A.sub.sample /A.sub.trypoin !.C.sub.trypoin.0.328 .V

A_(sample) : increase in absorbance/min of inhibited sample

A_(trypsin) : increase in absorbance/min of uninhibited trypsin

C_(trypoin) : trypsin concentration in the test mixture

V: (factor of dilution) predilution of the ETI solution

7 (factor of dilution)

    ______________________________________            C.sub.prot.            (protein concentration)                            Activity                                    Spec. activity    Protein (mg/ml)         (U/ml)  (U/mg)    ______________________________________    ETI (seeds)            0.81            0.71    0.88    recETI  0.83            0.89    1.07    ______________________________________

Result: The specific activity of recETI is 20% higher than the specificactivity of ETI isolated by classical methods from the seeds of E.caffra.

In further lots of recombinant ETI 1.2, 1.5 and 1.6 U/mg were forexample found as the specific activity.

EXAMPLE 4 Coupling recETI to CNBr-8epharoses®

170 mg purified recETI was dialysed against 0.05 M H₃ BO₃ /NaOH, pH 8.0,0.5 M NaCl (coupling buffer) and mixed with 7.5 g CNBr-Sepharose®(swollen overnight in 500 ml 1 mM HCl, then aspirated and suspended incoupling buffer). The suspension was incubated for 90 min at roomtemperature, aspirated and shaken overnight with 400 ml 0.1 M Tris/HCl,pH 8.0. The recETI-Sepharose® was aspirated and equilibrated with 0.7 Marginine/H₃ PO₄, pH 7.5.

EXAMPLE 5 Purification of a Recombinant Plasminogen Activator

54 mg recombinant plasminogen activator K2P (prepared according to WO90/09437 or U.S. Pat. No. 5,223,256) was applied to a recETI-Sepharosecolumn equilibrated with 0.7 M arginine/H₃ PO₄, pH 7.5. After washingwith equilibration buffer and with 0.3 M arginine/H₃ PO₄, pH 7.0 (fivecolumn volumes each time), it was eluted with 0.3 M arginine/H₃ PO₄, pH4.5. The plasminogen activator content in the eluate was determinedusing S 2288 as the substrate (Kohnert et al., Prot. Engineer. 5 (1992)93-100).

Result: The binding capacity of the recETI-Sepharose for asminogenactivator is 1.2 mg (corresponding to 0.63 MU) asminogen activator/mlrecETI-Sepharose.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 2    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 539 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 9..527    #/note= "Met only included inON:                   prokaryontic - # expression"    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1..8    #/function= "multiple cloning site"    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 528..539    #/function= "multiple cloning site"    #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #GTG GTG CAG AAC GGC      50AT GGT AAC GGC GAA             Met Val Leu Leu Asp G - #ly Asn Gly Glu Val Val Gln Asn Gly    #        10    - GGT ACC TAT TAT CTG CTG CCG CAG GTG TGG GC - #G CAG GGC GGC GGC GTG      98    Gly Thr Tyr Tyr Leu Leu Pro Gln Val Trp Al - #a Gln Gly Gly Gly Val    # 30    - CAG CTG GCG AAA ACC GGC GAA GAA ACC TGC CC - #G CTG ACC GTG GTG CAG     146    Gln Leu Ala Lys Thr Gly Glu Glu Thr Cys Pr - #o Leu Thr Val Val Gln    #                 45    - AGC CCG AAC GAA CTG AGC GAT GGC AAA CCG AT - #T CGT ATT GAA AGC CGT     194    Ser Pro Asn Glu Leu Ser Asp Gly Lys Pro Il - #e Arg Ile Glu Ser Arg    #             60    - CTG CGT AGC GCG TTT ATT CCG GAT GAT GAT AA - #A GTG CGT ATT GGC TTT     242    Leu Arg Ser Ala Phe Ile Pro Asp Asp Asp Ly - #s Val Arg Ile Gly Phe    #         75    - GCG TAT GCG CCG AAA TGC GCG CCG AGC CCG TG - #G TGG ACC GTG GTG GAA     290    Ala Tyr Ala Pro Lys Cys Ala Pro Ser Pro Tr - #p Trp Thr Val Val Glu    #     90    - GAT GAA CAG GAA GGC CTG AGC GTG AAA CTG AG - #C GAA GAT GAA AGC ACC     338    Asp Glu Gln Glu Gly Leu Ser Val Lys Leu Se - #r Glu Asp Glu Ser Thr    #110    - CAG TTT GAT TAT CCG TTT AAA TTT GAA CAG GT - #G AGC GAT CAG CTG CAT     386    Gln Phe Asp Tyr Pro Phe Lys Phe Glu Gln Va - #l Ser Asp Gln Leu His    #               125    - AGC TAT AAA CTG CTG TAT TGC GAA GGC AAA CA - #T GAA AAA TGC GCG AGC     434    Ser Tyr Lys Leu Leu Tyr Cys Glu Gly Lys Hi - #s Glu Lys Cys Ala Ser    #           140    - ATT GGC ATT AAC CGT GAT CAG AAA GGC TAT CG - #T CGT CTG GTG GTG ACC     482    Ile Gly Ile Asn Arg Asp Gln Lys Gly Tyr Ar - #g Arg Leu Val Val Thr    #       155    - GAA GAT TAT CCG CTG ACC GTG GTG CTG AAA AA - #A GAT GAA AGC AGC     52 - #7    Glu Asp Tyr Pro Leu Thr Val Val Leu Lys Ly - #s Asp Glu Ser Ser    #   170    #      539    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 173 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Met Val Leu Leu Asp Gly Asn Gly Glu Val Va - #l Gln Asn Gly Gly Thr    #                 15    - Tyr Tyr Leu Leu Pro Gln Val Trp Ala Gln Gl - #y Gly Gly Val Gln Leu    #             30    - Ala Lys Thr Gly Glu Glu Thr Cys Pro Leu Th - #r Val Val Gln Ser Pro    #         45    - Asn Glu Leu Ser Asp Gly Lys Pro Ile Arg Il - #e Glu Ser Arg Leu Arg    #     60    - Ser Ala Phe Ile Pro Asp Asp Asp Lys Val Ar - #g Ile Gly Phe Ala Tyr    # 80    - Ala Pro Lys Cys Ala Pro Ser Pro Trp Trp Th - #r Val Val Glu Asp Glu    #                 95    - Gln Glu Gly Leu Ser Val Lys Leu Ser Glu As - #p Glu Ser Thr Gln Phe    #           110    - Asp Tyr Pro Phe Lys Phe Glu Gln Val Ser As - #p Gln Leu His Ser Tyr    #       125    - Lys Leu Leu Tyr Cys Glu Gly Lys His Glu Ly - #s Cys Ala Ser Ile Gly    #   140    - Ile Asn Arg Asp Gln Lys Gly Tyr Arg Arg Le - #u Val Val Thr Glu Asp    145                 1 - #50                 1 - #55                 1 -    #60    - Tyr Pro Leu Thr Val Val Leu Lys Lys Asp Gl - #u Ser Ser    #               170    __________________________________________________________________________

We claim:
 1. A process for isolating a plasminogen activator from aprotein mixture, comprising the steps of:expressing, in a prokaryotic oreukaryotic cell, an exogenous nucleic acid which encodes a polypeptidewhich has the same specific inhibitory activity for tissue plasminopenactivator as a tissue plasminogen activator inhibitor DE-3 fromErythrina caffra, chromatographically purifying said polypeptide bymeans of an anion exchanger, cation exchanger or by means of a nickelchelate, immobilizing said polypeptide, binding a plasminogen activatorcontained in a protein mixture to said immobilized polypeptide,separating unbound components from the immobilized polypeptide, removingthe plasminogen activator from the immobilized polypeptide, andrecovering the isolated plasminogen activator, wherein said polypeptidehas an amino acid sequence according to SEQ ID NO:2.
 2. The processaccording to claim 1, wherein said polypeptide is immobilized on aninert carrier.
 3. A process for the production of a polypepuide whichhas the same specific inhibitory activity for tissue plasminogenactivator as a tissue plasminogen activator inhibitor DE-3 fromErythrina caffra, comprising the steps of:transforming or transfectingprokaryotic or eukaryotic host cells with an exogenous nucleic acidwhich encodes a polypeptide with the amino acid sequence of SEQ ID NO:2,culturing said host cells under conditions which result in theexpression of said polypeptide, chromatographically purifying saidpolypeptide by means of an anion exchanger, cation exchanger or by meansof a nickel chelate, and isolating said polypeptide, wherein saidpolypeptide has a specific inhibitory activity towards trypsin of 1.07U/mg and wherein said polypeptide has an amino acid sequence accordingto SEQ ID NO:
 2. 4. The process according to claim 3, wherein saidexogenous nucleic acid corresponds to nucleotides 9 to 527 of SEQ IDNO:1.
 5. The process according to claim 3, wherein said polypeptide isisolated by chromatographic purification on an anion exchanger, cationexchanger or on a nickel chelate column.
 6. An isolated and purifiedpolypeptide, wherein said polypeptide is obtainable by a processcomprising the steps of:transforming or transfecting prokaryotic oreukaryotic host cells with an exogenous nucleic acid which encodes apolypeptide according to SEQ ID NO:2, culturing said host cells underconditions which result in the expression of said polypeptide,chromatographically purifying said polypeptide by means of an anionexchanger, cation exchanger or by means of a nickel chelate, andisolating said polypeptide wherein said polypeptide has an amino acidsequence accordking to SEQ ID. NO:2.
 7. A recombinant polypeptideaccording to claim 6, wherein said polypeptide has the same specificinhibitory activity for tissue plasminogen activator as a tissueplasminogen activator inhibitor DE-3 from Erythrina caffra, and whereinsaid polypeptide has a specific activity towards trypsin of 1.07 U/mg ormore.
 8. The polypeptide according to claim 6, wherein said exogenousnucleic acid corresponds to nucleotides 9 to 527 from SEQ ID NO: 1.